Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson, Science, 1995, 267, 1456-1462).
Several stimuli can induce apoptosis, and recently, major advances have been made in understanding the signaling pathways downstream of these stimuli. The predominant pathway involves a proteolytic cascade orchestrated by a family of enzymes known as caspases (Thornberry, Br. Med. Bull., 1997, 53, 478-490).
An early event in apoptosis is the proteolytic cleavage of poly (ADP-ribose) polymerase or PARP, an enzyme involved in DNA repair, genome surveillance and integrity that is activated in response to environmental stress. The enzyme responsible for this cleavage is caspase 3, also known also known as apopain, CPP32 (Nicholson et al., Nature, 1995, 376, 37-43) and Yama (Tewari et al., Cell, 1995, 81, 801-809). Caspase 3 is a key pro-apoptotic protease that has been shown to cleave essential repair proteins (Casciola-Rosen et al., J. Exp. Med., 1996, 183, 1957-1964; Teraoka et al., FEBS Lett., 1996, 393, 1-6), huntingtin, the product of the Huntington's disease gene (Goldberg et al., Nat. Genet., 1996, 13, 442-449), amyloid protein precursor (Barnes et al., J. Neurosci., 1998, 18, 5869-5880), actin (Mashima et al., Oncogene, 1997, 14, 1007-1012) and a broad range of others (Tan and Wang, Trends Cell. Biol., 1998, 8, 116-120). Disclosed in PCT publication WO 96/25501 is a naturally occurring nucleic acid encoding Yama (a synonym for caspase 3), the Yama protein, antibodies that bind the protein and cell lines to produce the antibody (Dixit, 1996). The PCT publication, WO 96/33268 also discloses a purified caspase 3 enzyme under the name apopain. Antibodies and antisense molecules derived from synthetic DNA encoding caspase 3 molecules are also generally disclosed (Miller et al., 1996).
Like all caspases, caspase 3 is synthesized as a proenzyme and then cleaved, producing two subuints which reform the active enzyme (Nicholson et al., Nature, 1995, 376, 37-43).
Caspase 3 is involved in apoptosis induced by several agents including ceramide (Anjum et al., FEBS Lett., 1998, 439, 81-84), beta-lapachone (Shiah et al., Cancer Res., 1999, 59, 391-398), the Sendai virus (Bitzer et al., J. Virol., 1999, 73, 702-708), erythropoietin (Gregoli and Bondurant, Blood, 1997, 90, 630-640), granzyme B (Darmon et al., Nature, 1995, 377, 446-448), c-myc (Kangas et al., Oncogene, 1998, 16, 387-398), interferon gamma (Dai and Krantz, Blood, 1999, 93, 3309-3316), chemotherapeutic agents (Chen et al., Cancer Res., 1996, 56, 5224-5229) and peroxynitrite (Virag et al., Free Radic. Biol. Med., 1998, 25, 1075-1082). Furthermore, caspase 3 has been localized to apoptotic cells in several diseases including atherosclerotic plaques (Mallat et al., Circulation, 1997, 96, 424-428) and stomach (Hoshi et al., Anticancer Res., 1998, 18, 4347-4353), breast (Kurokawa et al., Oncol. Rep., 1999, 6, 33-37) and ovarian cancers (Chen et al., Cancer Res., 1996, 56, 5224-5229) as well as Fas-mediated apoptosis of hepatitis (Schlegel et al., J. Biol. Chem., 1996, 271, 1841-1844; Suzuki, Proc. Soc. Exp. Biol. Med., 1998, 217, 450-454). It has also been demonstrated that caspase 3 is activated in response to ischemia induced by cardiac arrest in hippocampal neurons (Gillardon et al., Brain Res. Mol. Brain Res., 1997, 50, 16-22) and co-localizes in apoptotic myocytes after ischemia reperfusion in rats (Black et al., J. Mol. Cell. Cardiol., 1998, 30, 733-742).
Caspase 3 deficient mice demonstrate lower than expected birth rate and size and die between one and three weeks of age. In addition, these mice exhibited severe abnormalities in brain development attributed to reduced neuronal apoptosis (Kuida et al., Nature, 1996, 384, 368-372).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of caspase 3.There are two naturally occurring viral protein products that inhibit caspase 3, CrmA, a serpin from cowpox virus (Tewari et al., Cell, 1995, 81, 801-809) and p35, a baculoviral product (Thornberry, Br. Med. Bull., 1997, 53, 478-490).
To date, strategies aimed at modulating caspase 3 function have involved the use of antibodies, molecules that block upstream entities such as the overexpression of bcl-2 (Monney et al., Biochem. Biophys. Res. Commun., 1996, 221, 340-345) and macromolecular and peptide inhibitors (Karanewsky et al., Bioorg. Med. Chem. Lett., 1998, 8, 2757-2762; Rodriguez et al., J. Exp. Med., 1996, 184, 2067-2072). Other inhibitors are disclosed in U.S. Pat. No. 5,798,442 (Gallant et al., 1998) and U.S. Pat. No. 5,834,228 (Becker et al., 1998) and in the PCT publications WO 99/18781 (Keana et al., 1999), WO 98/22098 (Granville et al., 1998) and WO 96/33209 (Gallant et al., 1996).
Consequently, there remains a long felt need for additional agents capable of effectively inhibiting caspase 3 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of caspase 3 expression. The present invention provides compositions and methods for modulating caspase 3 expression.